天津医药

天津医药

• 细胞与分子生物学 •    下一篇

基于双荧光素酶报告基因系统的人源SREBP1基因启动子的活性研究

杨念 1，李琛 2，李洪亮 2，郑燕 2，房树华 3，汪选斌 2△，赵雅玮 1△   

  1. 项目基金：国家自然科学基金面上项目（81874356）；湖北省卫健委重点项目（WJ2017Z023）；湖北医药学院自由探索基金资助 （2018YHKT01）；湖北省卫健委中医药科研青年人才项目（ZY2019Q004） 作者单位：1江苏大学附属句容医院药剂科（邮编212000）；2湖北医药学院附属人民医院肿瘤中心中药药理实验室，生物医药研究院，武 当特色中药研究湖北省重点实验室；3镇江市第一人民医院 作者简介：杨念（1991），女，硕士，药师，主要从事药理学研究 △通讯作者 汪选斌 E-mail：wangxb@hbmu.edu.cn；赵雅玮 E-mail：1241579758@qq.com
  • 出版日期:2019-09-15 发布日期:2019-09-15

Study on the activity of human SREBP1 promoter based on dual luciferase reporter system

YANG Nian1, LI Chen2, LI Hong-liang2, ZHENG Yan2, FANG Shu-hua3, WANG Xuan-bin2△, ZHAO Ya-wei1△   

  1. 1 Department of Pharmacy, Jurong Hospital Affiliated to Jiangsu University, Zhenjiang 212000, China; 2 Laboratory of Chinese Herbal Pharmacology, Oncology Center, Renmin Hospital, Biomedical Research Institute, Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei University of Medicine; 3 Zhenjiang First People’s Hospital
  • Published:2019-09-15 Online:2019-09-15

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摘要： 目的 构建固醇调节元件结合蛋白 1（SREBP1）启动子双荧光素酶报告基因表达载体，为研究针对 SREBP1靶点的天然活性抑制剂筛选奠定基础。方法 利用生物信息学软件，对SREBP1基因5′端上游5 000 bp序 列进行启动子预测与分析。应用 Gibson Assembly 方法构建启动子双荧光素酶载体，并将构建成功的 pGL3- SREBP1-pro重组质粒和内参质粒pRL-SV40共转染肝癌HepG2细胞并给予天然抑制剂大黄素，检测SREBP1转录活 性的抑制效果。结果 SREBP1基因5′端上游2 000 bp区域内有启动子特征序列，存在转录因子结合位点；重组质粒 经酶切及测序鉴定证实为阳性克隆，瞬时转染肝癌HepG2细胞后经双荧光素酶报告基因系统检测，所克隆的片段序 列具有启动子活性，该活性可被大黄素所抑制。结论 成功构建了pGL3-SREBP1-pro双荧光素酶报告基因表达载 体，并证实其活性，可为进一步用于针对SREBP1靶点的天然活性抑制剂筛选奠定

关键词: 固醇调节元件结合蛋白1, 转录启动子, 基因, 报告, 萤光素酶类, 大黄素, 蒽醌类, 生物信息学

Abstract: Objective To construct a dual luciferase reporter expression vector of sterol regulatory element-binding protein 1 (SREBP1) gene and to screen natural active inhibitors for SREBP1 targets. Methods The bioinformatics approaches were applied to predict and analyze the promoter region of SREBP1 from 5′ upstream 5 000 bp regulatory region. The dual luciferase reporter expression vector was constructed by Gibson Assembly method. The recombinant plasmid pGL3- SREBP1-pro and control plasmid pRL-SV40 were co-transfected into HepG2 cells. The inhibitory effects of the transcriptional activity of SREBP1 with or without natural active inhibitors were detected. Results There were promoter sequences and transcription factor binding sites in the 5′ upstream 2 000 bp region of SREBP1. The recombinant plasmid was confirmed as a positive clone by enzyme digestion and sequencing. The plasmid was transiently transfected in HepG2 cells to detect promoter activity of SREBP1 by dual luciferase reporter system, and transcriptional activity of SREBP1 was further verified by natural active inhibitors. Conclusion The pGL3-SREBP1-pro dual luciferase reporter expression vector is successfully constructed and its activity is confirmed, which can be further used for the screening of natural active inhibitors for SREBP1 targets.

Key words: sterol regulatory element binding protein 1, transcription initiation site, genes, reporter, luciferases, emodin, anthraquinones, bioinformatics